*2.4. Construction and Preparation of Recombinant DNA*

The SUMO (LdBPK\_080480) and SENP (LdBPK\_262070) coding sequences were amplified from *L. donovani* 1S genomic DNA using specific primer pairs (Table S1) that introduce restriction sites as indicated. PCR products were subsequently ligated into pCL2N [32], or derived plasmids pCL2N-3×HA (N-ter) and pCL2N-3×HA (C-ter), predigested with the cognate restriction enzymes. Plasmids were amplified in *Escherichia coli* DH5α and purified by CsCl density gradient ultracentrifugation as described previously [33].
